Cheese Whey Valorization via Microbial Fermentation (Lactic Acid Bacteria, Yeasts/Fungi, and Microalgae), Postbiotic Production, and Whey-Based Encapsulation Strategies

Abstract

Cheese whey, the major by-product of the dairy industry, poses an environmental challenge due to its high organic load but simultaneously represents a nutrient-dense matrix suitable for biotechnological valorization. This review synthesizes recent advances positioning whey as (i) a fermentation substrate for lactic acid bacteria, yeasts/fungi, and microalgae, enabling the production of functional biomass, organic acids, bioethanol, exopolysaccharides, enzymes, and wastewater bioremediation; (ii) a platform for postbiotic generation, supporting cell-free preparations with functional activities; and (iii) a food-grade encapsulating material, particularly through whey proteins (β-lactoglobulin, α-lactalbumin), which can form emulsions, gels, and films that protect biotics and bioactive compounds during processing, storage, and gastrointestinal transit. We analyze key operational variables (whey type and pretreatment, supplementation strategies, batch and continuous cultivation modes), encapsulation routes (spray drying, freeze-drying, and hybrid protein–polysaccharide systems), and performance trade-offs relevant to industrial scale-up. Finally, we outline future directions, including precision fermentation, mixed-culture processes with in situ lactase activity, microfluidics-enabled encapsulation, and life-cycle assessment, to integrate product yields with environmental performance. Collectively, these strategies reframe whey from a high-impact waste into a circular bioeconomy resource for the food, nutraceutical, and environmental sectors.

1. Introduction

The cheese industry generates whey as a by-product composed mainly of water, lactose, proteins, lipids, and minerals [1,2]. This effluent accounts for up to 90% of the milk volume used in cheese production, with an estimated global output of approximately 160 million tons annually [3]. Due to its high organic load and elevated biological and chemical oxygen demands (BOD and COD), whey disposal poses a major environmental challenge for the dairy industry [4,5]. Consequently, finding sustainable valorization strategies for this by-product has become a priority, particularly approaches that reduce its environmental impact while creating value-added products [6].

One promising strategy is the use of whey as a fermentation substrate and encapsulating ingredient. Owing to its abundance, nutritional composition, and low cost, whey serves as an excellent medium for microbial growth and biotransformation [7,8]. Fermentation is a well-established process in food biotechnology for producing proteins, bioactive peptides, and probiotics [9,10]. Whey provides a rich source of carbon and nitrogen that supports the growth of lactic acid bacteria (LAB), yeasts, and other microorganisms [11]. During fermentation, lactose consumption reduces whey’s BOD and leads to the formation of various metabolites, including organic acids, ethanol, and hydrogen, with potential applications across the food, pharmaceutical, and energy sectors [5,12].

In addition to fermentation, whey and its protein derivatives are effective encapsulating agents because they can form stable emulsions, gels, and films [13]. Encapsulation enhances the stability, bioavailability, and controlled release of bioactive compounds such as probiotics, enzymes, and nutraceuticals [14,15,16].

Overall, the valorization of cheese whey as a fermentation medium and encapsulating materials represents a sustainable strategy aligned with the principles of the circular economy [17]. Nonetheless, several challenges remain, including the optimization of fermentation parameters, the development of efficient encapsulation techniques, and the management of residual streams.

This review synthesizes recent advances in the valorization of cheese whey through microbial fermentation, including LAB, yeasts/fungi, and microalgae, together with emerging strategies for postbiotic production and whey-based encapsulation. We examine the microorganisms and process conditions involved, the types of bioactive metabolites and postbiotic preparations obtained, and the technological approaches enabling stabilization and delivery of biotic ingredients. Finally, we highlight key applications, opportunities, and remaining challenges shaping the role of whey revalorization in food, bioprocess, and biotechnology sectors. For clarity, the terms cheese whey and whey are used synonymously throughout in this review. While most examples focus on food and nutraceutical applications, whey valorization also supports broader biotechnological and environmental bioprocesses, which are included where relevant to provide a comprehensive perspective. The literature review included studies published between 2020 and 2025. Searches were conducted in the Scopus database using combinations of the keywords “whey”, “probiotic”, “yeast”, “fungi”, “microalgae”, “postbiotic”, and “encapsulation” within the title, abstract, or keywords. Studies were included if they addressed the fermentation of whey or whey-derived substrates, or the use of whey as a carrier or matrix for postbiotics or encapsulation systems.

2. Whey’s Composition and Process-Relevant Variability

2.1. Chemical Composition of Cheese Whey

Whey is the liquid fraction that remains after milk has been curdled during cheese production. It typically appears yellowish-green and represents 85–90% of the total milk volume, of which approximately 93% is water [6]. The solid fraction consists primarily of lactose (≈70%), proteins (≈14%), minerals (≈9%), and fats (≈4%). However, these proportions can vary depending on the type of cheese produced and the quality and origin of the milk [3].

There are two main categories of whey: sweet whey and acid whey. Sweet whey is produced from rennet-coagulated cheeses, where enzymes such as chymosin hydrolyze κ-casein, typically yielding a product with a pH around 6.5 [8]. By contrast, in acid whey, there is acidification, either through lactic fermentation or the addition of organic acids, which lowers the pH below 5 [8]. Despite these compositional differences, both types of whey exhibit high nutritional value, containing easily digestible proteins, fats, lactose, vitamins, and minerals [6]. Whey proteins have a biological value of 100, higher than casein, soy, or beef protein, and are particularly rich in sulfur-containing amino acids such as cysteine and methionine [18,19]. Additionally, whey supplies electrolytes such as sodium, potassium, and calcium, which can support microbial metabolism [12]. A further derivative, secondary cheese whey (SCW), is produced during additional processing steps such as creaming or the manufacture of ricotta [20].

From a biochemical perspective, whey provides an excellent nutrient profile for microbial growth. Lactose, its main carbohydrate, is a disaccharide composed of glucose and galactose that serves as a primary carbon and energy source for many fermentative microorganisms, particularly LAB, yeasts, and certain filamentous fungi [21]. Through enzymes such as β-galactosidase, these microbes hydrolyze lactose into monosaccharides that can enter glycolysis or alternative metabolic pathways. Whey proteins function as an essential nitrogen source, supplying amino acids and peptides required for microbial biosynthesis and cell proliferation [22]. Meanwhile, minerals such as calcium, magnesium, and potassium act as enzyme cofactors and contribute to osmotic balance and overall metabolic activity.

The high lactose and protein content of whey also contributes to its elevated BOD, typically 30,000–50,000 ppm, and COD, often reaching 60,000–80,000 ppm [6,23,24]. This dual nature makes whey both an environmental concern in its untreated form and a highly attractive substrate for biotechnological processes. Although transport and storage are challenging due to its volume and perishability, on-site concentration or drying can help reduce handling costs and logistical constraints. Overall, the biochemical composition of whey makes it a readily fermentable, nutrient-rich matrix that supports the growth of diverse microorganisms, setting the basis for the fermentation strategies discussed in the following sections.

2.2. Translating Whey Variability into Process Design Considerations

Although cheese whey composition is highly variable depending on its origin and processing, this variability can be translated into practical process design considerations related to pretreatment intensity, microorganism selection, and contamination control.

In practice, whey valorization strategies can be rationalized through a sequence of linked decisions: (i) whey type and composition (sweet vs. acid whey, lactose content, mineral load, pH); (ii) required pretreatment steps (pH adjustment, thermal or enzymatic treatment); and (iii) selection of microorganisms with appropriate metabolic capacity and robustness. For instance, high-lactose, low-salt sweet whey may be directly suitable for LAB or yeasts, whereas mineral-rich or saline dairy effluents favor halotolerant or marine-derived microorganisms.

Whey streams characterized by high BOD and COD values increase contamination pressure and often require thermal treatment or sterilization prior to fermentation [6]. Conversely, dilution or partial clarification can reduce organic load but may compromise overall process yields. Acid whey or streams with low pH and elevated mineral content may impose inhibitory conditions on conventional fermentative microorganisms, therefore shifting the process toward acid-tolerant or halotolerant strains and influencing pretreatment strategy selection.

Whey variability should not be regarded as a limitation but as a key determinant of process configuration, shaping pretreatment requirements, microorganism choice, contamination management, and overall process feasibility.

2.3. Implications for Performance Comparison Across Microbial Platforms

In the following sections, this review addresses whey fermentation across different microbial platforms, supported by comparative tables summarizing recent studies. Direct performance comparison among platforms is inherently constrained by the use of distinct evaluation metrics. LAB studies primarily report viable cell counts expressed as colony forming units (CFU) per g or mL, reflecting functional and regulatory requirements, whereas microalgae-related and heterotrophic protist studies emphasize biomass concentration, lipid content, or metabolite yield (g/L or % dry weight). In contrast, wastewater treatment-oriented studies prioritize removal efficiencies (% COD or BOD reduction). As a result, fully harmonized performance metrics across platforms remain impractical.

3. Whey Fermentation by Lactic Acid Bacteria

LAB comprise a diverse group of bacteria commonly represented in genera such as Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus. Although the term LAB lacks strict taxonomic designation, members of this group share key physiological traits: they are Gram-positive, non-spore-forming bacilli or cocci; predominantly aerotolerant anaerobes, and catalase- and oxidase-negative. Their central metabolism trait is the ability to convert carbohydrates primarily into lactic acid following either a homofermentative pathway via the Embden–Meyerhof–Parnas route or a heterofermentative metabolism yielding lactate together with ethanol or acetate and CO₂ in roughly equimolar proportions [25]. LAB have long been employed in dairy, meat, and plant fermentations due to their acidification capacity and their contribution to flavor, texture, and safety [26]. A key advantage of LAB is their intrinsic antimicrobial activity. Organic acid accumulation, primarily lactic and acetic acids, reduces pH to 3.5–4.5, inhibiting spoilage and pathogenic microorganisms. LAB also synthesize bacteriocins and reduce the redox potential, further limiting the growth of aerobic competitors [25].

Whey and its derivatives (sweet whey, acid whey, permeates, and SCW) contain high concentrations of lactose, peptides, and minerals that support LAB metabolism, although differences in pH, buffering capacity, salt balance, and residual lipids influence strain-specific performance [27,28,29]. Because whey contains fewer solids than milk, probiotic growth and survival may be reduced unless the matrix is supplemented with nitrogen sources (e.g., yeast extract), prebiotic fibers, or milk solids, or combined with mixed cultures [30].

Several LAB strains are considered probiotics capable of conferring health benefits when consumed at adequate levels [31,32]. Frequently used species in whey-based fermentations include Lactobacillus acidophilus, Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Limosilactobacillus reuteri, Bifidobacterium animalis, and others [33,34].

Table 1. Representative studies on lactic acid bacteria growth and functional fermentations in whey-based systems.

Microorganisms	Whey Matrix/Formulation	Conditions	Counts (Log CFU/mL)	Main Outcomes	Ref.
Mixed culture dominated by Lactobacillus delbrueckii	Cheese whey permeate + yeast extract (9 g/L)	CSTR; 45 °C; pH 6.0–6.5; HRT 1.5–10 h	74–99% L. delbrueckii biomass	Lactic acid 27.4 g/L/h (max); 70% yield	[30]
Lactococcus lactis BIOTEC007, Leuconostoc pseudomesenteroides BIOTEC012, Lentilactobacillus kefiri BIOTEC014, Lactobacillus acidophilus LA-3, Kluyveromyces lactis BIOTEC009	Pasteurized whey (100%) + skim milk, raspberry, inulin, carrageenan	30 °C × 24 h; 21 d at 4 °C	9.09 → 8.52 (21 d) Post-digestion: 8.23–8.52	GABA ↑ 1.3 → 4.65 mM; 40–45% retained post-digestion; good sensory acceptance	[35]
Lacticaseibacillus casei 1A, L Lacticaseibacillus paracasei 2A, Levilactobacillus brevis 4LB	90% fat-free whey + inulin + vitamins A, C, K	37 °C × 6 h; 7–10 d at 4 °C	7.0 → 8.0 (5 d) ≥ 107 (7 d)	Stable probiotic beverage; non-toxic (LD50); non-allergenic	[36]
Bifidobacterium breve BBR8, Bifidobacterium pseudocatenulatum M115	Whey protein isolate (10–20 g/L) + inulin or resistant starch	37 °C × 48–72 h; 30 d at 4 °C	8.5 → 6.5 (15 d)	RS > inulin for viability and sensory quality; peptides ↓ with storage	[37]
Streptpcoccus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus	Reconstituted whey + milk + sucrose + starch	43 °C × 5 h; storage 5 °C	Not reported	Improved weight gain, albumin, cholesterol (mice and children); 21 d shelf-life	[38]
Lacticaseibacillus rhamnosus RC007	100% cheese whey	37 °C × 16 h	8.0 (16 h)	Gut immunomodulation: ↑ IL-6, TNF-α, IL-10; ↑ goblet cells and IELs	[39]
Lactobacillus acidophilus LA-5, Bifidobacterium animalis BB-12	60% whey + yogurt + peppermint nanoliposomes	37 °C × 24 h; 60 d at 4 °C	10.4 → 5.7 (60 d)	Maintained viability ≤ 21 d; ↑ survival BB-12 > LA-5; viscosity ↓; syneresis ↑	[40]
Streptpcoccus thermophilus, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae	Parmigiano Reggiano whey (100%); spontaneous or NWS (2%)	42 °C; 6–72 h	LAB: 8.91; Yeasts: 1.78	Proteolysis ↑; ↑ ACE-, DPP-IV-, α-glucosidase inhibition; ↑ IPP and VPP; 49 bioactive peptides	[41]
Bacillus megaterium, Bacillus subtilis, Lactobacillus spp., Saccharomyces cerevisiae	13% whey broth + mineral salts	28 °C × 9 d	8.0 (28 d)	BOD ↓ 80%; ethanol 9.2 g/L; viable multi-strain probiotic	[42]
Kefir grain microbiota (Lactibacillus kefiranofaciens, Lentilactobacillus kefiri, yeasts and acetic acid bacteria)	SCW from ricotta	30 °C × 24 h; 10% w/v grains	Not specified	EPS 632.6 ± 30.8 mg/L; ↑ carbohydrate/protein ratio	[43]
Lactobacillus acidophilus LA-5, Bifidobacterium animalis BB-12	50% acid whey + milk/condensed milk	42 °C × 6–7.5 h; 21 d at 5 °C	≥8.0–8.9 (21 d)	Good sensory scores; acetaldehyde 0.3–1.6 mg/kg; textural differences between formulations	[44]
Mixed culture: Lactobacillus, Clostridium, Klebsiella	100% acid whey, delipidated	35 °C; pH 5.8; 5 d	LAB 86.6%; Clostridium 12.3%	H2: 44.5 ± 2.9 NmL/g-COD; hydrogen rate 1.9 NL/L·d; COD ↓ 24.6%	[45]

Arrows (↑ ↓) indicate increases or decreases in measured parameters. Abbreviations: ACE, angiotensin-converting enzyme; BOD, biological oxygen demand; CFU, colony forming units; COD, chemical oxygen demand; CSTR, continuous stirred-tank reactor; DPP-IV, dipeptidyl peptidase IV; EPS, exopolysaccharides; GABA, gamma-aminobutyric acid; H₂, hydrogen gas; HRT, hydraulic retention time; IELs, intraepithelial lymphocytes; IL-6, interleukin-6; IL-10, interleukin-10; IPP, isoleucine–proline–proline tripeptide; LAB, lactic acid bacteria; LD₅₀, median lethal dose; NWS, natural whey starter; RS, resistant starch; SCW, secondary cheese whey; VPP, valine–proline–proline tripeptide.

compiles a broad range of studies conducted in whey-based fermentation systems, illustrating the diversity of objectives, microbial consortia, and processing conditions explored to date. These works collectively highlight whey’s capacity to act as a multifunctional substrate for LAB growth, probiotic delivery, metabolite production, and innovative bioprocesses.

3.1. LAB Growth in Sweet Whey and Development of Functional/Probiotic Beverages

Extensive research has examined the use of whey as a base for probiotic and functional beverages. LAB typically reach levels ≥8 log CFU/mL in whey-based matrices when appropriately supplemented or combined with synergistic ingredients [46,47]. For example, strawberry whey beverages containing 0–80% whey fermented with L. acidophilus LA-14 maintained 8.68–8.83 log CFU/mL in all formulations [48], confirming whey’s suitability for supporting probiotic viability.

A multispecies consortium combining Lactococcus lactis, Lentilactobacillus kefiri, Leuconostoc pseudomesenteroides, L. acidophilus and Kluyveromyces lactis grew successfully in whey enriched with skim milk, raspberry and inulin. Cell densities remained ≥8 log CFU/mL over 21 days, while γ-aminobutyric acid (GABA), a neurotransmitter associated with stress modulation, increased from 1.3 to 4.65 mM with 40–45% post-digestion retention and a 58% consumer preference score [35]. This demonstrates the ability of whey to support both probiotic functionality and sensory-driven innovations.

Similar functional enhancements were observed in whey fortified with inulin and vitamins A, C, and K, which supported L. casei, Lacticaseibacillus paracasei, and Levilactobacillus brevis at ≥10⁷ CFU/mL for 7 days, with no signs of toxicity or allergenicity, confirming the safety and viability of fortified whey beverages [36]. Whey-derived ingredients also show promise: whey protein isolate (10–20 g/L) combined with inulin or resistant starch sustained Bifidobacterium breve and Bifidobacterium pseudocatenulatum at >8.5 log CFU/mL, with resistant starch providing superior stability and sensory attributes, highlighting whey derivatives as effective carriers for synbiotic formulations [37].

Beyond viability and technological performance, several studies have evaluated the physiological impact of whey-based probiotic beverages. Beverages fermented with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus improved weight gain, albumin levels, and cholesterol profiles in mice and children [38]. Fermentation of 100% cheese whey by L. rhamnosus RC007 enhanced gut immunomodulation, further supporting whey as a vehicle for targeted probiotic applications [39].

Additional technological approaches expand whey’s functional versatility. Nanoencapsulation strategies, such as formulations containing whey, yogurt, and peppermint nanoliposomes, maintained the viability of L. acidophilus LA-5 and B. animalis BB-12 over 21 days, although some textural changes occurred during storage [40].

Likewise, spontaneous fermentations of Parmigiano Reggiano whey using natural whey starters (S. thermophilus, Lactobacillus helveticus, L. delbrueckii subsp. lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae) enhanced proteolysis and generated bioactive peptides with cardiometabolic relevance, including well-known tripeptides such as IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro), noted for their antihypertensive and glucose-modulating potential [41].

Finally, co-culture strategies further broaden whey’s bioprocessing potential. Fermentation of a 13% whey broth with Bacillus megaterium, Bacillus subtilis, LAB, and S. cerevisiae produced a stable probiotic beverage after 28 days while reducing BOD by 80% and generating 9.2 g/L ethanol, demonstrating the feasibility of integrated probiotic–bioethanol systems [42].

3.2. LAB Activity in Secondary Cheese Whey, Acid Whey, and Mixed-Culture Systems

SCW, often underutilized, can also support robust microbial activity. Fermentation by kefir grain microbiota produced 632.6 ± 30.8 mg/L of exopolysaccharides (EPS) and increased the carbohydrate-to-protein ratio, evidencing SCW’s suitability for generating texture-enhancing and potentially prebiotic polysaccharides [43]. These findings underscore that lower-value whey streams can be effectively biotransformed into functional metabolites with direct applications in fermented foods and bioprocessing.

At a larger scale, industrial studies confirm that whey can sustain stable and productive mixed microbial communities. Whey permeate supplemented with yeast extract supported a mixed community dominated by L. delbrueckii in a continuous stirred-tank reactor, achieving lactic acid productivities of 27.4 g/L/h at 70% yield, and biomass compositions exceeding 90% L. delbrueckii [30].

Acid whey likewise supports LAB growth, despite its lower pH and compositional differences from sweet whey. Formulations with 50% acid whey and dairy ingredients supported the viability of L. acidophilus LA-5 and B. animalis BB-12 for 21 days and received favorable sensory evaluations [44].

Beyond food applications, mixed-culture fermentations broaden the potential of whey valorization into energy and biorefinery platforms. Delipidated acid whey used as substrate for dark fermentation by mixed cultures containing Lactobacillus, Clostridium, and Klebsiella generated hydrogen yields of 44.5 ± 2.9 NmL H₂/g COD and achieved 24.6% COD reduction, demonstrating the feasibility of integrating whey into renewable energy production pathways [45].

3.3. Limitations and Considerations for LAB Fermentations in Whey-Based Systems

Whey is a highly adaptable substrate for LAB, supporting probiotic growth, bioactive metabolite formation, mixed-culture fermentations, and diverse bioprocesses. However, several considerations must be addressed to optimize its use. Whey composition is highly variable, with fluctuations in lactose, minerals, and solids depending on cheese type and processing conditions; this affects LAB performance and may require supplementation with nitrogen sources or dairy solids. Its relatively low total solids content can also limit probiotic stability during storage unless fortified or combined with synergistic ingredients. Acid whey presents additional challenges due to its low pH, which can impair the viability of some LAB strains and alter sensory properties, making strain selection and buffering strategies important. Mixed-culture fermentations, while beneficial for functional outputs, require careful control to avoid undesirable dominance shifts or inconsistent metabolite profiles. Finally, sensory and physical stability issues, such as syneresis, reduced viscosity, or off-flavors, may arise in whey-based beverages, particularly during extended cold storage. Despite these limitations, whey remains a promising matrix for LAB-driven fermentations when its variability and strain-specific requirements are properly managed.

4. Whey Fermentation by Yeasts and Filamentous Fungi

Fungi, including yeasts and filamentous species, represent a metabolically versatile group of eukaryotic microorganisms with broad relevance in biotechnology. Their ability to secrete powerful extracellular enzymes, degrade complex organic substrates, and synthesize structurally diverse metabolites makes them strong candidates for whey valorization platforms [49,50]. While some fungi are opportunistic pathogens [51,52], many species are safely used in food, pharmaceutical, and industrial bioprocesses, contributing to fermentation, bioremediation, biofuel synthesis, and the generation of value-added metabolites [53,54]. Cheese whey, rich in lactose and nitrogenous compounds, provides a suitable substrate for many fungi capable of lactose assimilation, whether naturally or through genetic engineering, enabling applications that span from bioethanol and organic acids to single-cell oils, enzymes, pigments, and biocontrol agents [12]. Representative studies on fungal and yeast fermentations in whey are summarized in

Table 2. Representative studies on yeast and fungal growth in whey-based media and their biotechnological products.

Microorganism	Whey Matrix/Formulation	Conditions	Biomass/Productivity	Main Product/Purpose	Key Outcomes	Ref.
Kluyveromyces lactis Y-1564	Unprocessed cheddar sweet whey	30 °C, 72 h	Not reported	Ethanol	Ethanol 24.85 g/L; 99.4% lactose utilization; growth rate 1.89 h−1	[55]
Kluyveromyces marxianus DSM 5422	Delactosed whey permeate + urea; pH 5.1	Stirred bioreactor, 40 °C; batch/fed-batch/repeated-batch	24.3 g/L (Fe); 6–10 g/L (Fe-limited)	Ethyl acetate	Yield 0.347 g/g substrate; selectivity 97.8%; COD ↓ > 40%	[56]
Kluyveromyces marxianus WUT240	Whey permeate + L-phenylalanine + plant oils	Two-stage (biphasic + ethanol fermentation)	6.4–21.2 g/L (dry cell weight)	2-Phenylethanol + ethanol	2-PE 2.5 g/L; ethanol 75 g/L; BOD ↓ 92%; COD ↓ 46%	[57]
Alternaria sp. MH348917.1/Drechslera sp. MG582185.1	100% cheese whey (pH 6.0)	Submerged fermentation, 28 °C	Not reported	Single-cell oils	Lipids 3.22–4.33 g/L; PUFA up to 30%; antibiofilm > 90%; anticancer IC50 2.55–3.43%	[58]
Yarrowia lipolytica KKP 379	Acid whey (20–30%) + post-frying oil	Batch, 28 °C, 62–90 h	12–37 g/L	Single-cell oils and sterols	Lipids 38.8%; sterols 21.1 mg/g; UFA > 70%	[59]
Cutaneotrichosporon curvatus NRRL YB-775 & Papiliotrema laurentii NRRL Y-3594	Lactose media or SCW (56.5 g/L lactose)	Flask and fed-batch, 30 °C	>20 g/L biomass	EPS, lipids, mannitol	EPS up to 25.6 g/L; lipids 22%; COD ↓; TKN ↓	[60]
Geotrichum candidum NRRL Y-552/Penicillium corylophilum 802/Pleurotus ostreatus 3526/Penicillium restrictum 3381	Non-supplemented cheese whey	Aerated batch, 23 °C	8.3–16.9 g/L	Biomass and bioremediation	TN removal 54%; COD ↓ 43%; TP ↓ 34%; biomass rich in proteins and amino acids	[61]
Penicillium brevicompactum MUM 02.07	Whey + corn steep liquor; corncob support	SmFi, 23 °C, 12 d	Not reported	Natural pigments	Pigment yield +136% vs. synthetic medium; effective textile dyeing	[62]
Paecilomyces variotii VTT D-041019, Rhizopus oligosporus D-191691, Trichoderma reesei D-86271	Acid whey or potato cell fluid + glucose	Flask and 2 L bioreactor	20.7–36.5 g/L (d.w.)	Single-cell protein	Protein 16.9–26.8%; DF 23–29%; essential amino acids ↑	[63]
Mixed yeast culture, primarily Vanrija albida	Synthetic acid whey formulated to mimic acid whey	Batch mode; pH 3.5; high and low organic loading rates; high oxygen transfer efficiency	COD-use efficiency: 93% (high load) and 85% (low load)	Single-cell protein	Efficient COD removal; high oxygen transfer; material suitable for livestock feed with favorable amino acid profile	[64]
Kluyveromyces marxianus NRRL Y-1109 & Debaryomyces hansenii NRRL Y-1448	Whey + spent yeast + L-phenylalanine	Aerobic co-culture, 25 °C	K. marxianus: 1.0 log (CD/CD0); D. hansenii: 1.2 log	2-Phenylethanol	2-PE 1.84 g/L; 2.35× productivity; COD removal >99%	[65]
Yarrowia lipolytica RO3/Y3	Squacquerone cheese whey + 3% NaCl	Stirred tank, 24 °C	7.68–7.97 log CFU/mL	Adjunct cultures for cheese	↑ proteolysis; ↑ flavor volatiles; better sensory scores	[66]
Vishniacozyma victoriae	Whey-based medium	Cultivation + lyophilization	9.54 log CFU/mL	Biocontrol agent	Effective postharvest protection of pears against rot	[67]

Arrows (↑ ↓) indicate increases or decreases in measured parameters. Abbreviations: 2-PE, 2-phenylethanol; BOD, biochemical oxygen demand; CD/CD₀, colony density ratio; CFU, colony forming units; COD, chemical oxygen demand; DF, dietary fiber; d.w., dry weight; EPS, exopolysaccharides; UFA, unsaturated fatty acids; PUFA, polyunsaturated fatty acids; SmFi, submerged fermentation with mycelium immobilization; TN, total nitrogen; TP, total phosphorus; TKN, total Kjeldahl nitrogen.

.

4.1. Bioethanol Production from Whey by Yeasts and Filamentous Fungi

Bioethanol production remains one of the most thoroughly explored applications of fungal growth in whey. Although S. cerevisiae dominates industrial ethanol fermentations, its inability to metabolize lactose demands either genetic engineering strategies [68,69,70] or co-fermentations with lactose-consuming organisms. Naturally lactose-assimilating genera, including Kluyveromyces, Yarrowia, and some filamentous fungi such as Trichoderma and Aspergillus, can hydrolyze lactose extracellularly or import it into the cytosol for intracellular cleavage [12,70].

Industrial processes already employ K. marxianus, which tolerates high temperatures and low pH, although its ethanol yields remain generally lower than engineered S. cerevisiae under optimized conditions [71]. Several studies highlight the efficiency of K. lactis and K. marxianus grown in whey-based matrices. For instance, K. lactis cultivated in unprocessed cheddar sweet whey reached near-complete lactose utilization (99.8%) and produced 24.85 g/L ethanol with a remarkable 99.4% conversion efficiency, illustrating the strong potential of native lactose-consuming yeasts for energy-oriented biorefineries [55]. Similarly, K. marxianus cultivated in delactosed whey permeate under iron-limited conditions produced ethyl acetate with high selectivity (97.8%), a yield of 0.347 g/g substrate, and COD reduction exceeding 40%, demonstrating its suitability for volatile ester production in aroma and solvent industries [56]. Another K. marxianus strain grown in whey permeate with plant oils generated 2.5 g/L of 2-phenylethanol and 75 g/L ethanol in a two-step bioprocess, while simultaneously reducing BOD by 92% and COD by 46%, emphasizing its flexibility for multiproduct fermentations [57].

4.2. Yeasts and Fungi for Lipid, Sterol, and Single-Cell Oil Production

Beyond ethanol, several fungi efficiently convert whey-derived sugars into lipids and single-cell oils (SCOs), providing renewable feedstocks for biodiesel and nutraceuticals. Alternaria sp. and Drechslera sp. grown in 100% cheese whey produced 4.33 and 3.22 g/L lipids, respectively, with high polyunsaturated fatty acid content (up to 30%). Their extracts also exhibited strong antibiofilm (>90%) and anticancer activity (IC₅₀ 2.55–3.43%), suggesting multifunctional biotechnological relevance [58].

Yarrowia lipolytica, a well-established oleaginous yeast, demonstrated strong growth in whey–post-frying oil mixtures, reaching biomass levels of 37.44 g/L and lipid contents of nearly 39%, together with elevated sterol production (up to 21.08 mg/g biomass) and high unsaturated fatty acid proportions (>70%) [59]. These results highlight the suitability of whey as a carbon source for microbial lipid biorefineries and circular economy approaches integrating food waste streams.

Similarly, Cutaneotrichosporon curvatus and Papiliotrema laurentii cultivated in semi-defined lactose media or pretreated SCW achieved biomass levels exceeding 20 g/L, up to 25.6 g/L of EPS, and 22% intracellular lipids rich in oleic acid. Both processes reduced COD and total Kjeldahl nitrogen, demonstrating simultaneous wastewater treatment and high-value metabolite production [60].

4.3. Biomass, Enzymes, Pigments, and Functional Metabolites

Fungi grown in whey can also generate functional biomass and functional bioproducts. Non-supplemented whey supported the growth of Geotrichum candidum and Penicillium corylophilum, with biomass levels up to 16.9 g/L and removal of nitrogen (54%), COD (43%), and phosphorus (34%), demonstrating effective bioremediation alongside the generation of protein- and amino acid–rich fungal biomass [61].

Pigment production has also been reported. Penicillium brevicompactum cultivated in whey-based media supplemented with corn steep liquor produced fungal pigments with >136% higher yields than synthetic media, and the colored extracts were successfully applied to cotton and linen textiles without requiring mordant pretreatment, indicating potential for natural dye applications [62].

High-protein fungal biomass suitable for single-cell protein development was obtained using Paecilomyces variotii, Trichoderma reesei, and Rhizopus oligosporus, achieving biomass levels up to 36.5 g/L (dry weight) with 16.9–26.8% protein and 23–29% dietary fiber [63]. Similarly, acid whey-cultured Vanrija albida reduced oxygen demand and produced biomass with an amino acid profile adequate for livestock feed formulations [64].

4.4. Aroma Compounds, Enzyme-Rich Cultures, and Biocontrol Agents

Whey is also an efficient medium for producing aroma molecules and volatile compounds. Co-cultures of K. marxianus and Debaryomyces hansenii in whey supplemented with spent yeast generated 1.84 g/L of 2-phenylethanol, improving productivity by 2.35-fold over controls and removing >99% of COD [65]. Meanwhile, Y. lipolytica strains used as adjunct cultures in salted cheese whey produced desirable proteolytic and lipolytic activities, increased unsaturated fatty acids, and improved flavor volatile profiles (butanoic, hexanoic, and decanoic acids), earning positive sensory acceptance [66].

Biocontrol applications are also feasible: Vishniacozyma victoriae grown in whey reached 9.54 log CFU/mL, and the resulting biomass, after lyophilization, was successfully applied as a protective coating against postharvest pear rot [67].

4.5. Limitations and Considerations for Yeast and Fungal Fermentations

While whey is a promising substrate for diverse yeast and fungal processes, several constraints are specific to these organisms. Not all fungi possess the ability to metabolize lactose, restricting efficient growth to naturally lactose-assimilating species such as Kluyveromyces, Yarrowia, and certain filamentous fungi, or to strains that have been genetically engineered for this purpose. This narrows the range of species that can be directly used in whey-based bioprocesses.

In addition, whey often lacks the nitrogen levels and micronutrient balance required for optimal fungal performance. Many lipid-, SCO-, or enzyme-producing strains rely on supplemental nitrogen, trace minerals, or co-substrates to achieve desirable biomass levels and product yields. These nutritional adjustments are typically more demanding for fungi than for LAB.

Operational challenges also emerge during fungal fermentations. Filamentous species can form dense mycelial networks that limit mixing and mass transfer and may increase viscosity, while many yeasts require stringent aeration control to support metabolite synthesis such as 2-phenylethanol, ethyl acetate, or microbial lipids. These factors complicate scale-up and demand careful bioreactor design.

Contamination control is particularly important in fungal systems, since fungi are more vulnerable to competition from heat-resistant bacteria and wild yeasts commonly present in whey, which may survive standard thermal pretreatments. Ensuring appropriate hygienic and processing conditions is therefore critical to maintain culture purity and performance.

Despite these constraints, numerous studies show that yeasts and filamentous fungi can be highly effective biocatalysts for whey valorization when lactose assimilation, nutrient balance, and bioreactor conditions are properly aligned with the metabolic needs of each species.

5. Whey-Supported Cultivation of Microalgae

Microalgae, encompassing both eukaryotic microalgae and prokaryotic cyanobacteria, are versatile photosynthetic microorganisms capable of thriving in freshwater, marine, and wastewater environments [72]. Their biotechnological relevance has expanded rapidly due to their high nutritional value, rich diversity of bioactive metabolites, capacity to accumulate lipids and carbohydrates, adaptability to stress, and ability to remediate nutrient-rich effluents while fixing atmospheric CO₂ [49]. These attributes make microalgae strong candidates for the circular valorization of cheese whey, particularly through systems that couple wastewater bioremediation with the production of functional biomass for food, fuel, and pharmaceutical applications.

Early studies exploring whey–microalgae interactions focused primarily on nutrient removal, driven by the ability of microalgae to assimilate excess nitrogen, phosphorus, organic carbon (COD/BOD), and lactose from dairy streams [73]. More recently, this approach has evolved toward dual-purpose bioprocesses in which whey simultaneously serves as (i) a low-cost carbon source for microalgal growth and (ii) a substrate whose remediation increases environmental sustainability [74,75]. This coupling of bioremediation with biomass valorization defines the core of the emerging whey–microalgae circular bioeconomy.

5.1. Microalgae for Pigment, Metabolite, and Enzyme Production

A growing number of studies demonstrate that supplementing microalgal media with moderate concentrations of cheese whey can enhance pigment accumulation, metabolite synthesis, and enzymatic activities.

For example, Porphyridium purpureum cultivated in an artificial seawater medium containing 5.6% cheese whey (equivalent to 2.5 g/L lactose in the medium) under continuous light showed increases in phycoerythrin (+130%) and phycocyanin (+107%), demonstrating whey’s ability to stimulate the biosynthesis of high-value phycobiliproteins [76]. Similarly, Dunaliella salina grown with 20% whey at pH 7.5 in Loeblich medium exhibited a 39% increase in chlorophyll content and higher cell densities (from 2.10 ± 0.11 × 10⁷ to 2.70 ± 0.45 × 10⁷ cell/mL) [77], underscoring the stimulatory effects of organic components in whey on carotenoid-rich microalgae.

Other works highlight shifts in pigment profiles. Desmodesmus sp. grown in sterilized whey showed a significant rise in carotenoids (from 0.12 to 0.50 μg/mL) while chlorophylls decreased (from 6.75 to 1.64 μg/mL), suggesting metabolic rerouting under mixotrophic conditions [78].

Whey can also induce specialized enzymatic responses. Chlorella sorokiniana cultivated in whey under heterotrophic, dark conditions produced β-galactosidase with activities up to 0.090 U/mL, whereas no enzyme activity was detected in conventional BBM medium [79]. This demonstrates that lactose-rich whey can upregulate carbohydrate-active enzymes relevant to industrial processes.

5.2. Microalgae for Biodiesel and Biofuel Precursors

Another major application of whey–microalgae systems is the production of lipids and biofuel precursors. Oscillatoria sp. grown on a mixture of 25% whey and modified BG-11 medium increased biobutanol production from 2.6 to 4.2 g/L, showing the feasibility of coupling whey conversion with microbial biofuel synthesis [80]. A mixed community dominated by the cyanobacterium Leptolyngbya sp. and the microalga Ochromonas sp. generated lipids up to 14.8% of dry biomass when grown in whey-containing media (7.4–13.8%), yielding 124 mg/L of lipids suitable for biodiesel production while simultaneously removing up to 94% of whey’s organic load and >90% of nitrogen and phosphorus [81].

Likewise, Chlorella vulgaris cultivated in 50% whey improved lipid accumulation (from 71.43 to 80.90 mg/L), and the resulting fatty acid profile aligned with international biodiesel quality standards, confirming whey’s applicability as a carbon-rich substrate for algal biodiesel platforms [82].

5.3. Microalgae for Integrated Bioremediation and Biomass Valorization

Several studies demonstrate that microalgae can simultaneously remove nutrients from whey and convert the remaining carbon into valuable biomass, often under mixotrophic conditions [83,84,85,86,87,88,89,90,91,92,93,94].

Table 3. Representative studies on microalgae growth and bioremediation in whey-based media.

Microalgae Species	Whey Matrix/Concentration	Biomass Productivity	Contaminant Reduction	Cultivation Mode	Purpose	Main Outcomes	Ref.
Dunaliella tertiolecta	20% whey effluent (F/2 medium)	2.51 g/L (≈8 × control)	Lactose fully depleted (7 d)	Mixotrophic	Biomass and EPS production	↑ biomass (+37%), ↑ EPS (+30%), ↑ proteins (3×), ↑ sugars (3.5×), ↑ antioxidant activity; ↓ chlorophyll and carotenoids	[83]
Chlorella sp.	50–100% PCW or SCW; 1:1 MCW	1.55–1.76 g/L	COD ↓ 11,390–19,680 mg/L; TKN ↓ 626–1423 mg/L; TP ↓ 167–256 mg/L; lactose ↓ 15.9 g/L	Mixotrophic	Saline whey bioremediation and EPS-rich biomass	↑ pigments; ↓ growth in high-salinity SCW; ↑ attachment to whey solids (salt protection)	[84]
Chlorella vulgaris	100% neutralized whey (pasteurized)	0.95 ± 0.07 g/L	COD ↓ 55.5%, TN ↓ 53.0%, TP ↓ 35.3%	Mixotrophic	Treatment of high-load whey using UV-mutants	↑ adaptation; ↑ pollutant removal vs. wild strain; growth enhanced by UV + acclimation; biomass valorized for fertilizers/biofuel	[85]
Chlorella sorokiniana SAG 211/8k	Whey permeate (hydrolyzed lactose)	2.4 ± 0.2 g/L	COD ↓ 54%; carbs ↓ 90%; glucose ↓ 99%	Cyclic auto/heterotrophic	Biomass enhancement and bacteria control	↑ biomass (+54%); ↑ Yx/s (0.25 g/g); bacterial control achieved; ↑ lutein	[86]
Tetradesmus obliquus LCE-01 ± Cunninghamella echinulata	Simulated tertiary whey effluent	0.29–0.85 g/L	COD ↓ 75–77%; TN ↓ 70–74%; TP ↓ 66–70%	Semicontinuous	Improved tertiary treatment via algae–fungus consortium	↑ contaminant removal; ↑ biomass; ↑ pH stability; faster treatment; met EU standards	[87]
Neochloris oleoabundans UTEX 1185	35% Acid whey (MBB medium)	EPS: 380 mg/L	COD not quantified; lactose consumed	Mixotrophic (continuous light)	EPS with bioflocculant/antimicrobial activity	↑ EPS; ↓ growth (salt/pH stress); antibacterial activity; ↓ lipids and polyphenols	[88]
Graesiella emersonii MSCL 1718	20% concentrated whey permeate	1.69 ± 0.15 g/L	Partial lactose hydrolysis (↓ 3.88 g/L)	Mixotrophic	Biomass production and β-galactosidase assessment	↑ biomass; β-galactosidase 7.67 U/L; tolerated up to 40 g/L lactose; fast flocculation; stable pH	[89]
Chromochloris zofingiensis (CCAP 211/51)	10–50% whey wastewater (BBM)	3.86 g/L (10% WW)	TN ↓ 92.7%; COD ↓ 85.5%; lactose ↓ 99.9%	Mixotrophic	Wastewater treatment and astaxanthin/lipid production	↑ lipids (30.5%), ↑ astaxanthin (0.71 mg/g), ↑ carotenoids; biodiesel FA profile (C16–C18, 97%)	[90]
Graesiella emersonii MSCL 1711; Tetradesmus obliquus MSCL 1710	20–50% sweet or acid whey	0.29–0.30 g/L/day	Lactose ↓ 53%; β-galactosidase 51.9 U/L	Mixotrophic/heterotrophic	Biomass, lipids and lactose hydrolysis	↑ biomass; highest lipids in G. emersonii at 15 °C; optimal growth in sweet whey; intracellular β-galactosidase activity	[91]
Polyculture (Scenedesmus, Monoraphidium, diatoms)	1% whey permeate + landfill leachate	183.8 ± 87.7 mg/L/day	COD ↓ 497–524 mg/L/day; TN ↓ 21.7 mg/L/day; TP ↓ 3.0 mg/L/day	Mixotrophic (outdoor raceway)	Large-scale nutrient recovery and biomass	↑ biomass (5–9×); ↑ nutrient recovery; stable biochemical profile; no bacterial dominance	[92]
Chlorella vulgaris UTEX 265 (UV-mutant)	100% whey (neutralized, pasteurized)	0.89 ± 0.28 g/L	TN ↓ 3.7%; TP ↓ 17.1%; COD ↓ 3.1%	Mixotrophic	Full-strength whey bioremediation	↑ growth; biomass enriched in proteins, pigments, phenolics; coexistence with bacteria reduced N-removal	[93]
Nannochloropsis limnetica SAG 18.99	5% whey powder (DPBP medium)	1.06–1.36 g/L	TN ↓ >80%; P ↓ >80%; lactose ↓ 6 g/L	Mixotrophic	β-galactosidase production and nutrient removal	↑ β-galactosidase (40.8 U/L); ↑ TN and TP removal; stable algae–bacteria coexistence; reduced biomass in non-sterile cultures	[94]

Arrows (↑ ↓) indicate increases or decreases in measured parameters. Abbreviations: BBM, Bold’s Basal Medium; COD, chemical oxygen demand; DPBP, Defined Photoautotrophic Basal Medium; EPS, exopolysaccharides; EU = European Union; F/2; Guillard’s F/2 Nutrient Medium; FA, fatty acid; MCW; Mixed Cheese Whey; MBB; Modified Basal Broth; PCW; Primary Cheese Whey; SCW; Secondary Cheese Whey; TKN; Total Kjeldahl Nitrogen; TN, Total Nitrogen; TP; Total Phosphorus; UV; ultraviolet; UTEX, University of Texas Culture Collection of Algae; WW, Whey Wastewater; Yₓ/ₛ, biomass yield per substrate.

summarizes representative studies illustrating these integrated bioremediation and valorization strategies.

Dunaliella tertiolecta grown in 20% cheese whey achieved biomass levels ~8× higher than controls and increased EPS by 30%, while fully depleting lactose by day 7. Chlorella sp. cultivated in saline primary or secondary whey removed large quantities of COD, nitrogen, and phosphorus and produced EPS-rich biomass with improved pigment accumulation [83].

Other works show the potential of microalgae to tolerate high-strength whey streams despite elevated lactose and salinity levels and the presence of other pollutants, while maintaining robust growth, effective nutrient removal, and biomass valorization [84]. Ultraviolet (UV)-mutagenized C. vulgaris grown in undiluted whey achieved 55% COD reduction and produced pigment- and protein-rich biomass suitable for fertilizer or biofuel applications [85].

Cyclic cultivation strategies also enhance performance. Chlorella sorokiniana grown in ultrafiltration permeate under alternating light/dark cycles achieved +54% biomass compared with autotrophic controls, while consuming 90–99% of carbohydrates and maintaining bacterial contamination under control [86].

Microalgae–fungus consortia offer further gains: a co-culture of Tetradesmus obliquus and Cunninghamella echinulata increased contaminant removal (COD, total nitrogen, and total phosphorus) and nearly doubled biomass productivity, meeting EU discharge standards in only 3 days [87].

5.4. Microalgae-Related Heterotrophic Protists (Thraustochytrids and Oomycetes) for Whey Bioremediation and Lipid Valorization

Microalgae-related heterotrophic protists—particularly thraustochytrids and oomycetes—have emerged as alternative platforms for the valorization of cheese whey and dairy side-streams into high-value lipids. These organisms belong to the Stramenopiles lineage, which includes microalgae such as diatoms, but they are cultivated heterotrophically on organic substrates [95].

Thraustochytrids, especially Aurantiochytrium spp., are well-established producers of docosahexaenoic acid (DHA). Whey-derived streams have been used to cultivate Aurantiochytrium mangrovei, achieving DHA contents of 1.21 g/L content and biomass concentrations of 10.14 g/L following enzymatic hydrolysis of the side stream [96]. A subsequent techno-economic assessment comparing refined media with food-industry side streams, including dairy wastewater, highlighted that whey-based scenarios required hydrolysis and dilution steps, substantially increasing production costs [97].

Oomycetes such as Pythium irregulare represent a less explored but promising relevant group of lipid-producing protists. Cheese whey effluent from mozzarella production has been evaluated as a growth medium for eicosapentaenoic acid (EPA) production, following neutralization and thermal treatment to improve nutrient availability [95]. The whey-based media supported stable biomass growth, reflecting the high salinity tolerance of P. irregulare and its suitability for dairy effluent valorization.

Taken together, these studies demonstrate that whey-based fermentations can be extended to unconventional microbial platforms, expanding opportunities for lipid production and dairy by-product valorization.

5.5. Limitations and Considerations in Whey–Microalgae Systems

The performance of microalgae cultivated in whey-based media is strongly shaped by two operational variables: the concentration of whey in the culture medium and the selected cultivation mode. Whey concentration plays a central role, as higher proportions of this residue provide greater environmental benefits by reducing freshwater demand and increasing the volume of effluent treated. However, elevated concentrations of salts, organic load, and lactose can generate stressful conditions that inhibit microalgal growth. For this reason, many studies employ diluted whey—typically between 5% and 35%—to maintain suitable physiological conditions for biomass production [98]. To counteract inhibition at higher concentrations, strategies such as gradual acclimatization, whey sterilization, or the use of UV-mutagenized strains have been developed, resulting in improved tolerance and bioremediation efficiency [85,99].

The cultivation mode further modulates biomass accumulation and metabolite synthesis. Because whey inherently provides organic carbon, most microalgae grow under mixotrophic conditions, where photosynthesis is complemented by the uptake of organic substrates. Mixotrophic growth has been associated with high growth rates and biomass productivity, supported by enhanced carbon assimilation, cell division, and oxidative stress tolerance [100]. Under these conditions, increased nutrient removal is often observed across diverse strains and whey formulations [83,84,85,86,87,88,89,90,91,92,93,94]. Heterotrophic cultivation, relying exclusively on organic carbon in the absence of light, is less common but can be advantageous for targeted applications such as β-galactosidase production. In contrast, autotrophic cultivation—based solely on light and inorganic carbon—is seldom used in whey-based systems, as the presence of lactose and other organics naturally promotes mixotrophic or heterotrophic metabolism.

Overall, the interplay between whey concentration and cultivation strategy shapes not only growth performance but also the biochemical profile of the resulting biomass. Process optimization is therefore essential to maximize both bioremediation efficiency and the generation of high-value algal products.

6. Postbiotic Production from Whey

The production and characterization of postbiotics derived from whey have emerged as a promising research area. According to the current International Scientific Association for Probiotics and Prebiotics consensus, postbiotics are “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host” [101]. Unlike conventional whey fermentations aimed at generating viable microbial biomass, postbiotic-focused processes exploit whey as a substrate to produce bioactive metabolites, cellular components, or inactivated cells with documented physiological effects. Representative studies discussed in this section are summarized in

Table 4. Representative studies on postbiotic production using whey as substrate.

Microorganism/System	Whey Matrix/Formulation	Postbiotic Preparation	Purpose/Postbiotic Focus	Main Outcomes	Ref.
Lacticaseibacillus casei 01	Reconstituted whey + grape juice (50 g/100 g) + guar gum	Ohmic heating (8 V/cm; 95 °C, 7 min, 60 Hz)	Hypoglycemic functional beverage	↑ SCFAs, EPS, phenolic metabolites; antioxidant, anti-inflammatory and antimicrobial activity; hypoglycemic effects in vitro/in vivo	[102]
Kefir lactic acid bacteria	Whey + citrus pomace extract (polyphenol-rich)	Cell-free after bioconversion	Anti-obesity postbiotic	↑ Hesperetin; ↓ body weight gain, adiposity and triglycerides; ↑ UCP-1/PGC-1α; modulation of butyrate-producing microbiota	[103]
Lentilactobacillus kefiri DH5	Whey + 0.5% grape seed flour extract	Sonication + centrifugation + freeze-drying	Anti-sarcopenic postbiotic	↑ Muscle mass, grip strength; ↑ IGF-1, Atrogin-1; strong anti-inflammatory and myogenic gene regulation; synergy > whey or grape seed flour alone	[104]
Lactobacillus acidophilus LA-5 (postbiotic powders)	Whey + skim milk (12% w/w) + 1% peptone	Freeze-drying (–60 °C, 48 h, 0.065 mbar)	Yogurt fortification with LA-5 postbiotics	↑ Antioxidant activity (≈2× DPPH); ↑ acidity; ↓ syneresis; stable WHC; no negative effects on starter viability, rheology or sensory acceptance	[105]
Bifidobacterium lactis BB-12	Supplemented cheese whey (0–5% yeast extract; 30–45 °C; 12–36 h)	No inactivation (optimization of metabolite production)	Co-production of CLA, EPS and bacteriocins	Optimal: 38 °C, 28 h, 2.5% yeast extract → max CLA, EPS and BACs; temperature, yeast extract and time significantly influenced yields	[106]
Lactiplantibacillus plantarum PTCC1745	Whey, milk, or MRS as culture media	Heating (95 °C/5 min), sonication, centrifugation	Antimicrobial/antifungal postbiotics	Whey-based postbiotics as active as MRS; heating produced nano-postbiotics; antibacterial and antifungal activity retained; food matrices require pH adjustment	[107]
Lactiplantibacillus plantarum 299v, Lacticaseibacillus casei Shirota, Bifidobacterium animalis subsp. lactis BPL1	10% whey powder + minerals; whey + inulin (1–2%) or chia mucilage (1–2%)	Cell-free supernatant (centrifugation)	SCFA-rich and antioxidant postbiotics	Enhanced probiotic growth; 2% inulin → highest SCFAs (BPL1); L. plantarum & L. casei produced mainly lactic/acetic acids; ↑ antioxidant activity with fibers	[108]
Lactobacillus acidophilus LA-5	Whey (pH 4.5, autoclaved, +1% yeast extract) vs. MRS broth	Centrifugation + 0.45 µm filtration + freeze-drying	Comparison of whey- vs. MRS-derived postbiotics	Whey postbiotics richer in carboxylic acids; stronger antimicrobial activity; MRS-postbiotics showed higher antioxidant capacity	[109]

Arrows (↑ ↓) indicate increases or decreases in measured parameters. Abbreviations: BACs, bacteriocins; CLA, conjugated linoleic acid; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EPS, exopolysaccharides; IGF-1, insulin-like growth factor-1; MRS, de Man–Rogosa–Sharpe medium; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator-1 alpha; SCFAs, short-chain fatty acids; UCP-1, uncoupling protein-1; WHC, water-holding capacity.

.

6.1. Technological Approaches for Postbiotic Preparation

Postbiotic production requires two key stages: (1) fermentation to generate bioactive compounds and microbial structures, and (2) deliberate inactivation followed by processing to obtain stable preparations. Whey-based systems employ several technological strategies, each influencing the chemical profile and biofunctional properties of the final product.

Examples include thermal inactivation techniques such as ohmic heating, which achieves rapid microbial killing while preserving heat-sensitive compounds [102], and freeze-drying, widely used to stabilize postbiotic powders while maintaining antioxidant or anti-obesity effects [103,104,105]. Physical disruption methods such as sonication, centrifugation, and membrane filtration are commonly applied when producing cell-free supernatants enriched in key metabolites like EPSs, conjugated linoleic acid (CLA), short-chain fatty acids (SCFAs), and bacteriocins [106,107,108,109]. In certain cases, heat treatments have also been shown to produce nanoscale postbiotic structures with antimicrobial potential, expanding their applicability to functional foods or pharmaceutical systems [107].

Together, these approaches demonstrate the technological adaptability of whey as a substrate and its compatibility with food-grade processes aimed at producing safe, stable, and biologically active postbiotic ingredients.

6.2. Whey as a Substrate for Postbiotic Production

Recent studies demonstrate that whey efficiently supports the synthesis of core postbiotic metabolites by probiotic species such as L. casei, B. lactis, L. kefiri, and Lactiplantibacillus plantarum. For example, whey-based fermented beverages produced with L. casei 01 exhibited antioxidant, anti-inflammatory, antimicrobial, and hypoglycemic properties, driven by the generation of SCFAs, EPS, and phenolic metabolites [102]. Complementarily, metabolite optimization studies using B. lactis BB-12 in supplemented whey showed that CLA, EPS, and bacteriocins can be maximized through precise control of yeast extract supplementation, temperature, and fermentation time [106].

Hybrid fermentations that combine whey with plant-derived co-substrates broaden the spectrum of postbiotic metabolites. When whey was bioconverted with polyphenol-rich citrus pomace extract by kefir LAB, hesperidin was transformed into the bioactive hesperetin, yielding a postbiotic with strong anti-obesity effects and gut microbiota modulation in vivo [103]. Similarly, combining whey with grape seed flour extract and fermenting with L. kefiri DH5 produced a multifunctional postbiotic capable of mitigating muscle atrophy, improving muscle mass, strength, and myogenic gene expression [104].

Whey also supports the production of postbiotics based on inactivated cellular structures. Studies using L. plantarum PTCC1745 demonstrated that whey and milk can serve as effective, food-compatible media for producing antibacterial and antifungal postbiotics, including nanoscale structures generated through heat treatment [107].

Food applications further reinforce whey’s versatility. Freeze-dried L. acidophilus LA-5 postbiotics produced from whey–skim milk matrices enhanced antioxidant activity, improved water retention, and maintained sensory and rheological attributes when incorporated into yogurt [105]. Other investigations highlight that enriching whey with soluble fibers such as inulin or chia mucilage stimulates microbial growth and significantly increases SCFA yields and antioxidant capacity in cell-free postbiotic preparations from L. plantarum, L. casei, and B. lactis BPL1 [108]. Comparative studies between whey-derived and MRS-derived postbiotics show that whey supports the formation of distinct metabolite profiles, including higher levels of carboxylic acids and stronger antimicrobial effects, whereas MRS-derived preparations display greater antioxidant capacity [109].

6.3. Limitations and Considerations for Whey-Based Postbiotic Production

As discussed in earlier sections, whey streams exhibit substantial variability in chemical composition that directly influences fermentation kinetics and microbial metabolism. These compositional fluctuations become particularly critical in postbiotic processes, where the goal is to generate specific metabolites or cell-derived structures rather than viable biomass. A major challenge lies in the standardization of processing conditions. Postbiotic production requires complete microbial inactivation followed by downstream operations. While these technologies are effective, they can significantly alter metabolite stability, generate batch-to-batch variability, or introduce additional costs. For example, heat-based inactivation may degrade thermo-labile compounds, whereas mechanical disruption can fragment cellular structures inconsistently. Moreover, the need to maintain food-grade safety while processing large volumes of whey demands careful control of contamination risks, especially when whey is derived from diverse industrial settings. Another important consideration is scalability. Many studies employ laboratory-scale fermentations with optimized nutrient supplementation (e.g., yeast extract, fibers, plant extracts) that may not directly translate to industrial feasibility. The balance between enhancing metabolite yields and maintaining cost-effectiveness remains a central limitation, particularly when competing with established media.

Finally, the biological activity of postbiotics depends not only on fermentation and inactivation conditions but also on the intrinsic chemical complexity of whey. Interactions between peptides, organic acids, residual lipids, and polyphenols (in hybrid fermentations) may create highly heterogeneous preparations. This complexity complicates compositional characterization, hinders cross-study comparisons, and highlights the need for standardized analytical criteria for defining and quantifying whey-derived postbiotics.

7. Encapsulation of Biotics Using Whey

The previous sections demonstrated that whey could function as a versatile fermentable substrate for LAB, yeasts, fungi, and microalgae, as well as an efficient medium for generating postbiotics and metabolite-rich preparations. For these microbial products, whether viable cells, inactivated biomass, or purified metabolites—to be effectively incorporated into foods, supplements, or therapeutic formulations, stabilization is essential. Encapsulation technologies therefore represent a critical downstream step, and importantly, whey itself can also serve as an encapsulating material, offering an additional valorization route within whey-based bioprocesses.

The global interest in biotic ingredients continues to rise, supported by market projections for probiotics (USD 58.17 billion in 2021 and projected to grow at a 7.5% compound annual growth rate through 2030) [110], which demands technologies capable of preserving biological functionality throughout processing, storage, and gastrointestinal transit. Encapsulation addresses this challenge by entrapping probiotics, prebiotics, postbiotics, or other bioactive compounds within a protective matrix that shields them from heat, oxygen, moisture, and pH fluctuations [111,112]. By limiting exposure to such stressors, encapsulation helps maintain bioactivity over shelf life and enhances the survival of biotics during storage and digestion [13].

The performance of an encapsulation system depends largely on the physicochemical characteristics of the wall material, ideally food-grade, biocompatible, and able to form cohesive films, gels, or emulsions [113,114]. A wide range of biopolymers meets these requirements, including alginate, chitosan, gelatin, starch, gums, and whey proteins [114]. Each material offers distinct functional advantages: alginate forms gentle hydrogels suitable for live cell entrapment; chitosan contributes mucoadhesion and antimicrobial activity; gelatin and starch enhance mechanical strength and flexibility; while whey-derived proteins provide excellent emulsifying and film-forming properties, adding both structural stability and nutritional value to encapsulated systems [113,114].

7.1. Encapsulation Potential of Whey and Its Derivatives

Whey and its derivatives have gained considerable attention as encapsulating materials for microbial cells and biomolecules due to their low cost, wide availability, and multifunctional composition [115]. Their effectiveness is largely defined by their high protein content, which imparts excellent emulsifying, gelling, and film-forming capacities. These functional properties enable whey-based matrices to create cohesive microcapsule walls capable of protecting sensitive biotic ingredients during processing, storage, and gastrointestinal transit [112,113]. The resulting protein networks, stabilized through hydrophobic interactions, electrostatic forces, and disulfide bonds, also reduce oxygen permeability and moisture migration, two major causes of degradation in encapsulated systems [116].

Chemically, whey proteins possess structural attributes highly suitable for encapsulation. Their amphiphilic nature supports emulsion stability, while their ability to denature and cross-link under thermal or pH-induced conditions facilitates the formation of robust, semi-permeable membranes that withstand mechanical and thermal stress [117]. When blended with polysaccharides such as alginate, starch, or inulin, whey proteins form hybrid matrices with enhanced rigidity, heat tolerance, and controlled-release behavior, further improving protection and targeted delivery of encapsulated probiotics, postbiotics, or bioactive metabolites [112,113].

Whey contains several proteins that contribute differently to encapsulation functionality [118,119]. β-lactoglobulin, representing roughly 50–60% of total whey proteins, plays the predominant role due to its flexible globular structure, high surface activity, and strong film-forming capacity at oil–water interfaces, while α-lactalbumin accounts for 20–25% of whey proteins and contributes to gelation and thermal stability during matrix formation [120]. Immunoglobulins (approximately 10–15%) and lactoferrin (0.1–0.2%) add bioactive and antioxidant properties that further protect encapsulated cells or compounds, while bovine serum albumin (~5%) and glycomacropeptide (1–2%) offer additional stabilizing functions [121].

These combined properties explain why whey-derived systems, whether using whey protein concentrate (WPC) or whey protein isolate (WPI), have been successfully employed to encapsulate volatile flavors, lipids, and a broad range of probiotic microorganisms [121,122,123]. Their ability to form thin, cohesive films during technologies such as spray drying enables large-scale production of microcapsules with high retention of bioactive compounds and strong protection against oxidation and degradation [124]. Overall, the structural versatility and functional performance of whey proteins position whey as a valuable encapsulating agent within food and nutraceutical bioprocessing.

7.2. Encapsulation Methods Using Whey or Whey Derivatives

7.2.1. Spray-Drying

Spray drying is the most widely employed encapsulation technique in the food industry because it allows continuous, large-scale production at relatively low cost and provides good retention of viable microorganisms [125]. During the process, whey proteins denature and form cohesive films that act as protective microcapsule walls, reducing heat damage and oxidative stress.

Whey has been successfully employed as an encapsulating material in several formulations. When bovine and buffalo cheese whey were used to encapsulate Lactiplantibacillus pentosus ML 82 and L. plantarum ATCC 8014, survival rates reached approximately 95% after drying, with cell counts remaining above 7 log CFU/g after 90 days of storage. Bovine whey produced more compact, low-moisture capsules, whereas buffalo whey resulted in higher moisture content and slightly reduced viability, demonstrating how protein composition influences capsule stability [126]. Similarly, cheese and ricotta whey were effective matrices for encapsulating L. paracasei ATR6. Cheese whey generated smaller particles (~5.3 µm) with lower moisture (8.6%) and >78% survival, while ricotta whey produced larger, moister capsules (13.5%) and a >47% process yield, again underscoring the role of protein concentration in encapsulant performance [127].

Spray drying also shows enhanced performance when whey is combined with pre-concentrated substrates or polymer blends. Goat whey concentrate obtained through block freeze concentration enabled B. animalis ssp. lactis BB-12 to achieve encapsulation yields of 86.9–87.4% and ~9.5 log CFU/g after drying, with minimal losses during refrigerated storage (0.10–0.17 log reduction at 4 °C over 60 days). Incorporating inulin further improved powder solubility, highlighting the benefits of composite matrices [128].

Although spray drying is efficient and industrially scalable, the associated thermal and dehydration stress can compromise microbial survival if not properly optimized. Whey proteins mitigate these limitations by enhancing heat buffering and moisture retention within the microcapsule structure, contributing to improved stability and viability during and after processing.

7.2.2. Freeze Drying

Freeze drying is a low-temperature dehydration method that removes water by sublimation, allowing for excellent preservation of microbial structure and viability. Although energy-intensive and slower than other techniques, it is particularly suitable for formulations requiring high survival rates and long-term stability [129]. When camel and cow whey proteins were used to encapsulate Pediococcus acidilactici S30-4C, survival reached approximately 89%, with camel whey providing superior thermal tolerance—up to 99% survival at 50 °C for 5 min—and improved protection during simulated gastrointestinal digestion. The encapsulated cells also retained α-glucosidase and dipeptidyl-peptidase IV inhibitory activities, demonstrating that whey-based matrices can safeguard both viability and metabolic functionality [130].

Comparable results were obtained with alginate–WPC systems used for a probiotic starter culture: freeze-dried carriers showed higher survival and better solubility during storage than their spray-dried counterparts, confirming the strong preservation capacity of low-temperature dehydration [131].

While freeze drying provides exceptional stability and functional retention, its operational cost and long processing cycles limit large-scale commercial use. Nonetheless, it remains the preferred technique for high-value probiotic and postbiotic preparations where maintaining bioactivity is essential.

7.2.3. Hybrid and Composite Encapsulation Systems

To address the limitations of single-material encapsulation systems, recent research has focused on hybrid matrices that combine whey proteins with polysaccharides or carbohydrate-based carriers. These composite systems leverage the emulsifying and film-forming capacity of whey proteins while incorporating rigidity, heat resistance, and controlled-release behavior provided by complementary polymers. As a result, hybrid encapsulation improves mechanical stability, protects bioactives during environmental stress, and enhances performance under gastrointestinal conditions.

A representative example is the encapsulation of L. reuteri TF-7 using WPI blended with nanocrystalline starch. Spray-dried microcapsules achieved >90% encapsulation efficiency and ~9 log CFU/g after drying, while retaining 71% viability at 63 °C and 56% after gastrointestinal simulation, substantially outperforming free cells and maintaining bile salt hydrolase activity [132].

Similarly, Lactobacillus fermentum K73 encapsulated using either a sweet-whey–maltodextrin blend or a whey-based culture medium, followed by spray- or freeze-drying, showed that powders derived from the whey medium provided the highest gastrointestinal protection. Viability losses were only 0.23–0.38 log CFU/g in 1% fat milk, compared with ~5.5–5.6 log CFU/g in water, highlighting the synergistic compatibility between whey matrices and dairy environments [133].

Further evidence comes from mixed-material systems such as alginate–WPC carriers. These formulations achieve >80% encapsulation efficiency and maintain >7 log CFU/g during storage and fermentation. Spray-dried versions preserved up to 89% viability under gastrointestinal conditions, whereas freeze-dried powders showed slightly higher survival overall, confirming that hybrid systems can balance industry-scale feasibility with biological robustness [131].

The main advantage of hybrid systems lies in their tunability: combining whey proteins with polysaccharides increases capsule strength, moisture resistance, and thermal protection, addressing whey’s inherent sensitivity to heat and mechanical stress. Additional improvements can be achieved through cross-linking or enzymatic modification, which optimize barrier properties and enable targeted release tailored to specific food or nutraceutical applications.

8. Regulatory Frameworks and Safety Aspects in Whey Valorization

The application of whey-derived probiotics, postbiotics, and encapsulated ingredients in food systems is governed by regulatory frameworks that differ across regions. In the United States, microorganisms and microbially derived products intended for food use are commonly assessed under the Generally Recognized As Safe (GRAS) framework, whereas in the European Union, the Qualified Presumption of Safety (QPS) approach is applied by the European Food Safety Authority [134,135]. Currently, only a limited number of well-characterized LAB species are included in these lists, which may restrict the direct food application of non-conventional microorganisms.

In addition, novel postbiotic preparations or ingredients obtained through unconventional microbial platforms may fall under Novel Food regulations in the European Union, requiring additional safety and toxicological assessments prior to market authorization [136]. These regulatory considerations are particularly relevant when extending whey valorization strategies beyond traditional LAB toward microalgae-related protists or newly encapsulated bioactive compounds. Consequently, regulatory status and safety requirements should be considered early in process design to ensure translational and commercial feasibility.

Beyond ingredient-level approval, bioprocess-related safety regulations play a critical role in whey-based fermentations. Whey and dairy effluents are characterized by high organic load and susceptibility to microbial contamination, necessitating strict compliance with Good Manufacturing Practices, Hazard Analysis and Critical Control Points systems, and hygienic process design throughout fermentation, downstream processing, and encapsulation steps [137]. Together, regulatory approval and process safety considerations represent key constraints that shape the industrial implementation of whey valorization strategies.

9. Conclusions and Perspectives

The valorization of cheese whey as a fermentation substrate, postbiotic source, and encapsulating material highlights its strategic relevance within circular bioeconomy frameworks. Across LAB, yeasts, fungi, and microalgae, whey consistently supports microbial growth and the synthesis of valuable metabolites, while simultaneously enabling wastewater bioremediation and nutrient recycling. Furthermore, whey-based systems facilitate the production of postbiotics with beneficial properties, demonstrating that this dairy by-product can be transformed into a platform for high-value functional ingredients.

However, the literature reviewed in this work reveals several bottlenecks that limit industrial translation, including pronounced compositional variability of whey streams, trade-offs between productivity and contamination risk, sensitivity of many microorganisms to mineral load and pH, and increased costs associated with pretreatment and downstream processing. In response to these constraints, advancing whey bioprocessing will require greater process control, standardization, and predictive capacity. Precision fermentation approaches directly address the variability and inhibition challenges identified in whey-based systems by enabling improved lactose utilization, tighter metabolic regulation, and tailored metabolite profiles. In parallel, metabolic engineering and multi-omics tools support strain optimization by elucidating microbial responses to complex dairy matrices. Machine learning and data-driven modeling offer promising frameworks for predicting fermentation dynamics, optimizing co-substrate ratios, and designing robust microbial consortia capable of handling whey’s compositional variability. In parallel, emerging approaches such as multistage fermentation, adaptive laboratory evolution, and mixed cultures with in situ enzymatic hydrolysis (e.g., lactase-producing strains) have the potential to unlock new routes for producing biohydrogen, volatile compounds, and single-cell protein.

Encapsulation strategies based on whey proteins and hybrid matrices with polysaccharides will further expand the applicability of whey-derived biotics. Techniques such as spray drying, microfluidics, and freeze-drying enable the stabilization, controlled release, and integration of fermented whey products into food, nutraceutical, and biomedical formulations. Continued progress in polymer–protein hybrid materials may also support biodegradable packaging solutions enriched with functional properties.

Despite these opportunities, challenges remain. Compositional variability, high moisture content, and seasonal effects demand improved pretreatment methods (e.g., ultrafiltration, concentration, and demineralization). At the same time, industrial adoption will depend on pilot-scale validation, techno-economic analyses, and rigorous life cycle assessment to quantify environmental impacts and demonstrate sustainability advantages over conventional substrates. Overall, future research should prioritize: (i) the genetic and physiological improvement of microbial strains; (ii) the integration of precision fermentation and AI-driven process optimization; (iii) the development of stable, functional encapsulated systems; and (iv) comprehensive environmental and economic evaluations.

By addressing these areas, cheese whey can be fully consolidated as a versatile, renewable, and high-impact resource for sustainable innovation across the biotechnological, food, and environmental sectors.